ADCs are specific therapeutic monoclonal antibodies (mAb) chemically linked to cytotoxic payloads. The design is aimed to provide selective delivery of cytotoxic payloads to tumors and to exert tumor targeted killing effects. For an ADC to be successful, it needs to bind to its target antigen on tumor cells, internalize via receptor-mediated endocytosis and be transported to the lysosome where subsequent intracellular processing of the ADC will release the biologically active drug.

Prior to the making of an ADC, an appropriate antibody clone with optimal binding and internalization into the cells needs to be selected. Here we describe three in vitro characterization techniques to screen for various antibodies targeting glycosylphosphatidylinositol (GPI)-linked cell membrane protein. These in vitro characterization techniques assess both anti-proliferation as well as the kinetics of binding and internalization by imaging cancer cells. To understand the biological relevance of these in vitro activities, an in vitro/in vivo comparison was explored. Overall, a mAb that has improved binding and prolonged internalization kinetics may yield improved efficacy and durability when conjugated to toxic payloads.